Aquatic Botany 91 (2009) 213–218 Contents lists available at ScienceDirect Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot Asexual life history by biflagellate zoids in Monostroma latissimum (Ulotrichales) Felix Bast a,*, Satoshi Shimada b, Masanori Hiraoka a, Kazuo Okuda a a Graduate School of Kuroshio Science, Kochi University, 2-5-1 Akebono, Kochi 780-8520, Japan b Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo 112-8610, Japan ARTICLE INFO ABSTRACT Article history: Monostroma latissimum (Kuetzing) Wittrock is a monostromatic green alga of commercial importance in Received 16 December 2008 Japan. Here we report the serendipitous discovery of asexually reproducing specimens collected from Received in revised form 26 May 2009 Usa, on the Pacific coast of Kochi Prefecture, south-western Japan. Zoids were found to be biflagellate and Accepted 24 June 2009 negatively phototactic. Germination of settled zoids was observed to follow erect-filamentous ontogeny Available online 1 July 2009 similar to that of the previously reported sexual strain. Moreover, the newly discovered asexual strain had identical sequences of nuclear encoded ITS (Internal Transcribed Spacer) region to that of the sexual Keywords: strain. On the basis of this finding, we postulate that the ITS sequences may have been maintained in Internal Transcribed Spacer these conspecific strains despite the evolution in sexuality. Relationships were investigated among M. Life history Phylogeny latissimum and other monostromatic taxa within the class Ulvophyceae using ITS sequences in order to Seaweed understand relative phylogenetic position of this species. Taxonomy ß 2009 Elsevier B.V. All rights reserved. Thallus ontogeny 1. Introduction In this report, we present results of our life history and molecular studies to aid in understanding the identity of the marginal Monostroma (Chlorophyta, Ulvophyceae), a multicellular thal- populations of monostromatic green algae naturally occurring in loid green algal genus, consists predominantly of sexually a low saline habitat near Uranouchi Inlet, Tosa Bay, Kochi, Japan. reproducing dioecious species. The name ‘Monostroma’ is derived Monostroma latissimum (Kutzing) Wittrock (Wittrock, 1866), an from the Greek root: mono (single) and the Latin root: stroma intertidal seaweed, grows luxuriantly in this inlet from autumn (layered), reflecting its thallus architecture consisting of single through spring (Bast et al., 2009a). The life cycle of this species is layer of cells (Bold and Wynne, 1985). This genus was originally diplo-haplontic, with macroscopic gametophytic and microscopic characterized by Thuret (1854) as thalli forming leafy monostro- sporophytic generations (Kunieda, 1934; Tatewaki, 1969; Hua et al., matic blades 2–30 cm (or more) in length. Many taxa were 2004; Bast et al., 2009a). Secondary sex ratio in this species is subsequently added into this genus, all based upon Thuret’s reported to be 1:1 throughout the period of occurrence (Bast et al., original characteristic; i.e., fronds solely made-up of horizontally 2009b). As this species has previously been known to reproduce only arranged single layer of cells. A plethora of taxonomic revisions on sexually, we did not expect to find an asexual reproduction at our this genus – beyond the scope of the present report – resulted in study site. We also have sequenced nuclear encoded rDNA Internal the construction of many new genera to accommodate all of the Transcribed Spacer (ITS1) sequences from the new strain of monostromatic green algae. Recently, Hayden and Waaland (2002) Monostroma and previously identified sexually reproducing M. suggested that the single cell-layered thallus morphology appears latissimum to characterize sequence divergence between the two to have a polyphyletic derivation, as hinted by the completely strains. This molecular marker was chosen because ITS sequences different ontogenies of species with similar thallus morphology. are available for a large group of marine green algae and has a high Summary of the key taxonomic characters used to diagnose seven degree of variance even between very closely related algal taxa putative monostromatic genera: Monostroma, Protomonostroma, (Woolcott and King, 1998). Morphological and developmental Gayralia, Ulvaria, Kornmannia, Ulvopsis and Capsosiphon based on characteristics, combined with partial ITS sequences, had been life history, thallus ontogeny and other characteristics hitherto found to be useful in resolving species-level identity within the class known are listed in Table 1. Ulvophyceae (Hiraoka et al., 2003). 2. Materials and methods * Corresponding author. Tel.: +81 90 2899 5075; fax: +81 888448314. Thalli of monostromatic green algae were collected from an E-mail address: [email protected] (F. Bast). intertidal zone at a fresh water stream, near its confluence to the 0304-3770/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2009.06.006 214 F. Bast et al. / Aquatic Botany 91 (2009) 213–218 Table 1 Summary of taxonomic characters used to diagnose genera: Monostroma, Ulvopsis, Gayralia, Protomonostroma, Kornmannia, Ulvaria and Capsosiphon. Genus Life history Thallus ontogeny Zoid release Zoid ultrastructure Monostroma Heteromorphic alternation of leafy gametophytes Filament-blade/ Without pores Biflagellate gametes, quadriflagellate and cyst-like sporophytes filament-sac-blade zoospores. Eye-spot present Gayralia Non-alternation of generations (monomorphic asexual) Filament-sac-blade Without pores Biflagellate zoids. Eye-spot present Protomonostroma Heteromorphic alternation of generations (asexual). Filament-blade Without pores Quadriflagellate zoids. Eye-spot absent One generation is leafy and other is unicellular cyst Ulvopsis Heteromorphic alternation of leafy gametophytes and Disc-sac-blade Through pores Biflagellate gametes and asexual zoids, cyst-like sporophytes. Rarely non-alternating quadriflagellate zoospores. Eye-spot present (monomorphic) asexual Kornmannia Heteromorphic alternation of microscopic disc-like Disc-sac-blade Through pores Biflagellate gametes and quadriflagellate gametophytes and leafy sporophytes. Non-alternating zoospores. Quadriflagellate asexual zoids. (monomorphic) asexual also present Eye-spot absent Ulvaria Isomorphic alternation of generations Filament-sac-blade Through pores Biflagellate gametes and quadriflagellate zoospores. Eye-spot present Capsosiphon Heteromorphic alternation of tubular gametophytes and Filament-tube Through pores Biflagellate gametes and quadriflagellate cyst-like sporophytes; Rarely isomorphic alternation of zoospores. Asexual reproduction by generations and non-alternating (monomorphic) asexual quadriflagellate zoids or aplanospores. Eye-spot present in gametes Based on Gayral (1964); Bliding (1968) and Tatewaki (1969). N.A. stands for information not available. inlet (Uranouchi Inlet) in Usa, Kochi Prefecture, Japan (33.43N (293 bp for both strains) were performed as in Shimada et al. 133.44E), on April 2007. Salinity levels at the sampling site (2003). All phylogenetic analyses were conducted in Geneious were observed to be fluctuating substantially with a yearly average computer software, version 4.6 (available at www.geneious.com). of about 17 psu, while that at the inlet (10 m downstream) Sequences were aligned first using the built-in Geneious Aligner of was about 28 psu (Bast et al., 2009a). After washing in tap water, the same software, then edited by eye. ITS1 sequences of 10 related thalli were placed individually in BD-Falcon 12-well cell culture taxa were obtained from Genbank and included in the alignments. plates (BD Biosciences, Bedford, MA) with 2–3 mL modified Alignments are available from first author upon request and from enriched seawater (West and McBride, 1999) and cultured at Treebase (http://www.treebase.org/treebase). Phylogenetic ana- 15 8C under cool white fluorescent light with an irradiance of lysis using maximum likelihood (ML) algorithm was conducted 70 mmol photons mÀ2 sÀ1 and 12:12 h LD (light:dark) photoperiod using PhyML plug-in version 2.4.5 (Guindon and Gascuel, 2003) to induce maturity. Previously identified male and female inside Geneious, with starting tree generated by BioNJ. Substitu- gametophytes of the sexual strain of M. latissimum (Kuetzing) tion bias was modeled by the general time-reversible model (Yang, Wittrock collected from the inlet on March 2007 were also grown 1994a) with invariable sites (Hasegawa et al., 1985) and rate in the similar culture conditions. heterogeneity was modeled using the gamma distribution method Every 4 d the medium was changed in the culture plates. After (Yang, 1994b) with four discrete rate categories and a single shape two weeks, culture dishes were placed at 25 8C to facilitate the zoid parameter (alpha). Heuristic searches were performed with tree release. The size and number of flagella of zoids released from bisection-reconnection, MULTREES and steepest descent options in mature thalli were observed under a microscope (ECLIPSE E200, effect. A total of 1000 bootstrap replicates were performed under Nikon, Tokyo, Japan) and photographs were taken using a digital ML criterion to estimate branch support (Felsenstein, 1985). camera (COOLPIX4500, Nikon, Tokyo, Japan). Zoids/gametes were Bayesian posterior probabilities to indicate statistical support for collected using their phototactic response in autoclaved sea-water interior branches were calculated using MrBayes plug-in version 3 (Kawai et al.,